Hepatitis C is a severe hepatic disease caused by hepatic C virus (HCV). About 170 million people have been infected with HCV all over the world and over 40 million have been infected in China. About 50˜85% of the HCV-infected individuals will subsequently develop into chronic hepatitis and about 10˜15% will develop into hepatocirrhosis. So HCV is of huge harm to human health and lays a heavy burden on our society.
HCV diagnosis has been developed greatly since Choo et al managed to clone the HCV gene in 1989. At present, there are three main approaches to detecting Hepatitis C: detecting the HCV RNA by polymerase chain reaction (PCR); detecting the HCV antigens using a monoclonal antibody; and detecting the HCV antibody with Enzyme Linked Immunosorbent Assay (ELISA) and Western blotting, etc.
The ELISA reagent for detecting an HCV antibody has developed into the third generation. The primary Elisa-1 kit was developed by Ortho Clinical Diagnostics Inc. (U.K.) in 1990, using the HCV NS4 recombinant antigen C100-3 provided by Chiron (USA). Unfortunately, the Elisa-1 kit has rather unsatisfactory sensitivity and specificity. Thereafter, the Elisa-2 kit took the place of the Elisa-1 kit. The Elisa-2 kit was developed by Ortho Clinical Diagnostics Inc. (U.K.) and Abbott (USA) respectively, using antigens such as CORE, NS3 and NS4. The Elisa-2 kit has its sensitivity and specificity improved. It is found that the positive rate is 92˜95% when the Elisa-2 kit is applied to the chronic hepatitis C patients and the average window period of HCV seropositive conversion is shorted from 16 weeks to 10 weeks due to the application of CORE and NS3 antigens. Further, the Elisa-3 kit is developed and produced by Ortho Clinical Diagnostics Inc. (U.K.), Abbott (USA) and Sanofi (France) in the lead, using antigens such as CORE, NS3, NS4 and NS5. It is found that the sensitivity thereof is increased to about 97% and the average window period of HCV seropositive conversion is shorted to 7˜8 weeks when the ELISA-3 kit is used to detect HCV in blood donors and high risk groups.
Elisa has such advantages that it can be easily automated, conveniently operated and well reproduced in diagnosis. Moreover, it is low in cost and helpful in analysis and storage of the experimental data. Nowadays, commercial reagents that are employed all over the world for screening the HCV antibody are mainly Elisa-2 and Elisa-3 kits developed with the indirect method. Despite the improvements for three generations, the Elisa reagents are still quite disadvantageous as shown by the false negative, false positive, and uncertain results that mainly arise from the inherent defects of the indirect reagents in methodology. Specifically, the disadvantages are as follows:
1. Poor specificity. The indirect method applies labels of non-specific recognition to the second antibody, which leads to low specificity.
2. Low sensitivity. Because of poor specificity, the concentrations of the coating antigens and the labeled second antibody in the kits have to be lowered, the amount of the specimens to be detected has to be reduced, and the specimens have to be diluted in duplication, which thereby lowers sensitivity.3. Long window period. The common second antibody is the anti-IgG antibody that can only detect the IgG components in the antibody but cannot detect other types of antibody, especially the IgM antibody in the early period, which lowers sensitivity and prolongs the window period.
At present, 70% of the post-transfusion hepatitis cases are infected with Hepatitis C after blood transfusion, and 80□90% of the Hepatitis C-infected individuals fall within the group of post-transfusion infection, which results to a large degree from the above-mentioned defects of the current indirect reagents. Therefore, there is a need to develop an HCV antibody diagnostic reagent of high sensitivity and high specificity.
Double-antigen sandwich method substitutes a labeled antigen for a labeled second antibody in the indirect method, thereby overcoming the defects of the indirect method methodologically. Although it is widely applied to antibody detection nowadays, the rather advanced double-antigen sandwich method is still not employed in the kit for screening of the HCV antibody for two main reasons:
1. The Activity Loss Following the Labeling of HCV Protein
Presently labeling the antigen directly with a label such as peroxidase horseradish (HRP) is the dominant enzyme labeling method, but some problems arise when this method is applied to the labeling of HCV antigen. Firstly, the NS3 antigen as a main antigen epitope segment of HCV, is an antigen strongly dependent on its conformation; when labeled by a “flexible” label with a high molecular weight such as peroxidase horseradish or alkaline phosphatase, it varies greatly in conformation, and its active epitopes are difficult to expose. As such, the activity of labeled NS3 antigen will be substantially decreased. Secondly, the main active region of CORE antigen as another main antigen epitope segment of HCV contains lots of Lysine which are critical to the activity of CORE, and the free amino-group in the side chain of Lysine is often used as the target for label reaction (such as labeling HRP by NalO4 oxidization). Accordingly, the activity of CORE antigen is greatly decreased after labeling, and as a result the sensitivity of the kits produced is even poorer than that of indirect methods, which may leads to higher possibilities of mis-diagnosis.
2. The High Background
The CORE antigen has a strong homologous and heterogenous binding capacity in vitro or in vivo (Matsumoto, M. etc., Virology, 218:P 43-51, 1996; Kunkel, M. etc., Virology, 294: P 239-245, 2002; Majeau, N. etc., Journal of General Virology, 85: P 971-981, 2004)). In the double-antigen sandwich method, the labeled antigen (the secondary antigen) can bind homologously with the coated antigen (the primary antigen), which leads to high background of the kit.
By now there are lots of reports about the reagent for detecting the HCV antibody by the double-antigen sandwich method at home and abroad, but each is subjected to some defects mainly as follows:
The method introduced in the patent application of the publication No. CN 1548958A firstly took no account of the importance of the size of heterologous protein for maintaining activity of HCV antigen, particularly NS3 antigen, hence failed to screen the pontine protein, i.e., did not use the pontine protein having a molecular weight as small as possible to ensure the activity of HCV antigen so as to further ensure sensitivity of the kit. Secondly, the method did not add a certain proportion of sulfhydryl reagents (like β-mercaptoethanol or dithiothreitol, etc.) or denaturants (like sodium dodecyl sulfonate) (see U.S. Pat. No. 6,270,960 and U.S. Pat. No. 6,261,764 for more details) into a coating liquid or add a certain proportion of sulfhydryl reagents into a blocking liquid, which prevented the coating antigen from showing its activity completely, thereby lowering sensitivity of the kit. Thirdly, the method did not add a certain proportion of sulfhydryl reagents into the dilution liquid of the secondary antigen, which prevented the secondary antigen from showing its activity completely, thereby lowering sensitivity of the kit. Fourthly, the method failed to put forward a solution to the problem of high background caused by various binding tendencies of CORE antigens. In addition, in the examples of the patent application, the protein expressed by the empty vector pET-32a(+) contained His Tag, so that the polyclonal antibody labels thereof also included anti-His Tag antibody labels; however, since its coating antigen contained His Tag, the anti-His Tag antibody labels could combine directly with the coating antigen to develop color, so that all sera, either negative or positive, would present a positive reaction.
Although the method introduced by Baochang Lee et al (Journal of Experimental Hematology, 12(3): P 359-362, 2004) researched into HCV antigen biotinylation, the study was carried out merely on the level of antigen, without posing concrete solutions to the above-mentioned problem that might be confronted in the development of the HCV antibody diagnostic reagents of the double-antigen sandwich method. Moreover, the method proposed in the article for biotinylating HCV protein using primitive biotin-[acetyl-CoA carboxylase] synthetase of Escherichia coli in vivo had rather low efficiency in biotinylation, only 10% or so (Paul A. S. et al, Nucleic Acids Research, 26(6): P 1414-1420, 1998; Tsao, K. L. et al, Gene, 169: P59-64, 1996). To eliminate as much as possible the influence on sensitivity by competition of, when reacting as the secondary antigens, non-biotinylated antigens with biotinylated antigens, said biotinylation method had to separate the biotinylated antigens, which lowered the yield rate of biotinylated antigens and increased difficulty and cost of subsequent purification.
Although patent applications U.S. Pat. No. 6,096,319, U.S. Pat. No. 6,270,960 and U.S. Pat. No. 6,306,579 mentioned the HCV double-antigen bridge-sandwich method, they posed no substantive solution to the above-said difficulties. In fact, these patent appliations only set forth theories to apply various double-antigen sandwich diagnostic methods that were already rather mature in other fields to HCV diagnosis.
In the HCV double-antigen bridge-sandwich method disclosed in patent application U.S. Pat. No. 6,613,530, the antigens (including primary antigen and secondary antigen) were conjugates of HCV protein and carrier protein. Protein carriers (BSA, 69 KD; β-Gal, 465 KD; Bovine-Fab, 75 KD) with a larger molecular weight were only applicable to coupling short peptides of HCV, while the conformation-dependant HCV antigen, particularly NS3 segment antigen, had its activity obviously lowered when this method was employed, which thereby affected sensitivity of the kit.
The double-antigen sandwich method involved in the article of Cheng Cao et al (Chinese Journal of Medical Laboratory Technology, 19(4): P 205-207, 1996) was just an initial attempt to transplant double-antigen sandwich diagnostic methods that were already rather mature in application to other fields directly into HCV diagnosis, labeling HRP directly onto free lysine of unengineered HCV protein without considering the above characteristics of HCV protein, which greatly reduced activity of the labeled antigens, whereby the kits had the severe disadvantage of false negative results.
Using a kit for detecting an HCV antibody developed with the double-antigen sandwich method, this invention can not only fundamentally overcome the diverse disadvantages of the indirect method from the methodological perspective, but also solve the technical problems existing in the present double-antigen sandwich kit. That is, the present invention managed to develop a kit for detecting an HCV antibody applicable to clinical settings, which provides a more favorable tool for screening HCV.